Immortalization of cells including neuronal cells

ABSTRACT

The instant invention provides methods for immortalizing cells. The invention further provides immortalized cell lines, e.g., neuronal cell lines, and methods of using these cell lines in screening assays.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/671,865, filed 15 Apr. 2005, the entire contents of which areincorporated herein by reference.

GOVERNMENT SUPPORT

The following invention was supported at least in part by NIH Grants RO1NS43991 and PO1 MH70056. Accordingly, the government may have certainrights in the invention.

BACKGROUND OF THE INVENTION

Neuropathic pain, also referred to as a chronic pain, is a complexdisorder resulting from injury to the nerve, spinal cord or brain. Thereis evidence that nerve fibers in subjects with neuropathic pain developabnormal excitability, particularly hyper-excitability, Zimmerman (2001)Eur J Pharmacol 429 (1-3):23-37. Although the American Pain Societyestimates that nearly 50 million Americans are totally or partiallydisabled by pain, there are currently very few effective, well-toleratedtreatments available (Wetzel et al. (1997) Ann Pharmacother 31(9):1082-3). Indeed, existing therapeutics cause a range of undesirableside effects primarily due to the difficulty in developingsmall-molecule drugs capable of specifically targeting thereceptor/channel of choice.

Many, relatively common clinical conditions are associated withneuropathic pain (Berger A, et al. (2004) J Pain 5:143-149).Traditionally, combinations of tricyclic antidepressants oranti-epileptics along with analgesics have been used to treatneuropathic pain (reviewed in (Mendell J et al. (2003) N Engl J Med348:1243-1255). However, treatment of neuropathic pain is oftenunsatisfactory; persistent neuropathic pain affects quality of life andlead to significant morbidity. In recent years with the identificationof the receptor for capsaicin (Caterina M J et al. (1997) Nature389:816-824; Caterina, M et al. (2000) Science 288:306-313), neuropathicpain research has directed its attention to identification of drugs thatinterfere with the transient receptor potential vanilloid receptor 1(TRPV1) physiology. Primary effort has focused on antagonists that blocknociceptive pain sensation at the receptor level but so far, no drug hasreached clinical use (Caterina M J et al. (1997) Nature 389:816-824 andCaterina, M J et al. (2000) Science 288:306-313)

Previous attempts at identifying TRPV1 antagonists have usednon-neuronal cell lines expressing recombinant TRPV1 and the calciumflux induced by capsaicin as an outcome measure for high throughputscreening (HTS) (Caterina M J et al. (1997) Nature 389:816-824;Caterina, M J et al. (2000) Science 288:306-313). Although these cellsexpressing recombinant TRPV1 may be useful, a nociceptive sensoryneuronal cell expressing TRPV1 might be more relevant because thenon-neuronal cell lines may lack the appropriate intracellular signalingpathways associated with and downstream of TRPV1 in nociceptive sensoryneurons. In order to generate tools for a more rational approach to drugscreening for neuropathic pain, it would be useful to have animmortalized DRG sensory neuronal line with nociceptive properties. Todate, attempts to immortalize neuronal cell lines have achieved littlesuccess.

Likewise, the ability to generate immortalized cell lines using cellsthat have been historically difficult to immortalize would be beneficialin the efforts to develop novel therapeutics for the treatment ofdisease and illness.

Although neuronal cell lines have been generated in the past these weremostly from embryonic tissues and were derived from progenitor or stemcells (see, e.g., Bernard J (1989) Neurosci Res, 24:9-20, Evrard (1990)PNAS, 87:3062-6, Redies J (1991) Neurosci Res 30:601-15). Also, atemperature sensitive mutant T antigen has been used to immortalizeneuronal populations, but the efficiency of this technique has been verylow (Eves (1994) Brain Res 656:396-404).

Accordingly, the need exists for effective and reliable methods ofimmortalizing cells that scientists have not had success inimmortalizing with currently available methods, e.g., neuronal cells.

SUMMARY OF THE INVENTION

The instant invention is directed to methods for making immortalizedcell lines from cells that are historically difficult to immortalize,e.g., neuronal cells. The inventors of the instant application havediscovered a novel method for making stable immortalized cells, e.g.,neuronal cells.

Accordingly, in one aspect the instant invention provides, methods forgenerating an immortalized human cell comprising introducing into a cella DNA segment encoding an oncogene, selecting for a cell containing theDNA segment, and introducing hTERT into the selected cell, therebygenerating an immortalized cell.

In one embodiment, the DNA segment is contained in a plasmid. In anotherembodiment, hTERT is contained in a plasmid.

In another embodiment, the neuronal cells are selected from a groupconsisting of neuronal cells from the brain, neuronal cells from thespinal cord, dorsal root sensory ganglia, dorsal root ganglia neuron andautonomic ganglia. In a specific embodiment, the neuronal cell is, forexample, a nociceptive dorsal root ganglion neuron.

In another embodiment, the cell is a glial cell, e.g., an astrocyte,oligodendrocyte or a Schwann cell.

In another embodiment, the methods further comprises contacting theimmortalized cell with an agent that causes differentiation and/or axonelongation. In specific embodiments, the agent cyclic AMP or an analogthereof or an agent that increases intracellular cAMP levels. In aspecific embodiment, the cAMP analog is forskolin.

In another aspect, the invention provides methods of producingimmortalized neuronal cells comprising introducing a DNA segmentencoding an oncogene into a neuronal cell, selecting for neuronal cellsthat contain the DNA segment, introducing hTERT into the selected cells,and selecting for cells that contain hTERT, thereby producingimmortalized neuronal cells.

In one embodiment, the DNA segment is contained in a plasmid. In anotherembodiment, the hTERT is contained in a plasmid.

In another embodiment, the neuronal cells are selected from a groupconsisting of neuronal cells from the brain, neuronal cells from thespinal cord, dorsal root sensory ganglia, dorsal root ganglia neuron andautonomic ganglia. In a specific embodiment, the neuronal cell is, forexample, a nociceptive dorsal root ganglion neuron.

In another embodiment, the oncogene is selected from the groupconsisting of Ras, Myc, Raf, and large T-Antigen. In one particularembodiment, the oncogene is the large T-antigen, e.g., the SV40 largeT-antigen.

In another embodiment, the hTERT is contained in a plasmid.

In another embodiment, the methods further comprises contacting theimmortalized cell with an agent that causes differentiation. In specificembodiments, the agent cyclic AMP, an analog thereof, or an agent thatincreases intracellular cAMP levels. In a specific embodiment, the cAMPanalog is forskolin. In a related embodiment, the forskolin allows theimmortalized neurons to differentiate and extend axons.

In another embodiment, the immortalized nociceptive dorsal root ganglionneurons maintain the biochemical and electrophysiological properties ofprimary neurons. In a related embodiment, the nociceptive dorsal rootganglion neurons express a capsaicin receptor, TRPV1, GDNF-receptor,NGF-receptor, or a sodium channel. In a specific embodiment, thenociceptive dorsal root ganglion neurons express capsaicin receptorTRPV1. In another related embodiment, the nociceptive dorsal rootganglion neurons respond to capsaicin by elevating intracellular calciumflux or generate action potentials when polarized.

In another embodiment, the immortalized nociceptive dorsal root ganglionneurons express one or more axonal markers, e.g., neurofilament or βIIItubulin.

In another aspect, the invention provides methods of producingimmortalized dorsal root ganglion neuronal cell line comprising,introducing a plasmid comprising an SV40 large T-antigen into dorsalroot ganglion cell, selecting dorsal root ganglion cells that containthe plasmid, introducing hTERT into the selected cells, and selectingfor cells that contain hTERT, thereby producing immortalized dorsal rootganglion neuronal line.

In a related embodiment, hTERT is contained in a plasmid.

In another aspect, the invention provides immortalized nociceptivedorsal root ganglion neurons.

In another embodiment, the immortalized nociceptive dorsal root ganglionneurons maintain the biochemical and electrophysiological properties ofprimary neurons. In a related embodiment, the nociceptive dorsal rootganglion neurons express a capsaicin receptor, TRPV1, GDNF-receptor,NGF-receptor, or a sodium channel. In a specific embodiment, thenociceptive dorsal root ganglion neurons express capsaicin receptorTRPV1. In another related embodiment, the nociceptive dorsal rootganglion neurons respond to capsaicin by elevating intracellular calciumflux or generate action potentials when polarized.

In a specific embodiment, the invention provides immortalizednociceptive dorsal root ganglion neurons comprising an oncogene andhTERT. In exemplary embodiments, the oncogene is selected from the groupconsisting of Ras, Myc, Raf, and large T-Antigen. In one particularembodiment, the oncogene is the large T-antigen, e.g., the SV40 largeT-antigen.

The invention further provides methods for screening for modulators ofneuronal cells. These modulators are useful in, for example, thetreatment and prevention of pain. In one embodiment, the inventionprovides high throughput methods of screening using the immortalizedcells produced by the methods of the invention.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-EC depict immortalized DRG neuronal cells extend neurites,express neuronal markers and generate action potentials afterdifferentiation. Phase contrast images of undifferentiated (A) anddifferentiated (B) 50B11 cells show extension of axons 4 hours afterdifferentiation with forskolin. After differentiation, 50B11 cells stainwith anti-neurofilament (C) and anti-βIII-tubulin antibodies. DAPIcounterstain shows nuclei. Scale bar=20 μm. A representative actionpotential is seen in a differentiated 50B11 cell after application of adepolarizing current (E).

FIGS. 2A-C depict 50B11 neuronal line expresses markers of smalldiameter sensory neurons. Immunofluorescence images of 50B11 cellsstained with fluorescently tagged IB4 (A) and anti-CGRP (B). Nuclei arecounterstained with DAPI. Scale bar=20 μm. Changes in expression of mRNAfor p75, Trk-A, c-ret and GFRa-1 in the presence of forskolin (FSK), NGFand GDNF compared to baseline levels of undifferentiated 50B11 cells(C). (n=6-8/group; error bars denote standard error of mean; *=p<0.05compared to baseline; **=p<0.05 FSK+GDNF versus FSK+NGF)

FIGS. 3A-C depict 50B11 neuronal line expresses nociceptive markers andrespond to capsaicin. Immunofluorescence image of 50B11 cells stainedwith anti-TRPV-1 antibody (A). Nuclei are counterstained with DAPI.Scale bar=25 μm. Fold change in TRPV-1 mRNA in response todifferentiation and neurotrophic factor treatment is seen (13).(n=6-8/group; error bars denote standard error of mean; *=p<0.05compared to baseline; **=p<0.05 PSK+GDNF versus FSK+NGF) Measurements ofintracellular calcium levels of undifferentiated 50B11 with vehiclecontrol treatment (red line), after differentiation with forskolin with(green) and without (blue) capsazepin pretreatment (C). Single arrowindicate the time at which capsazepin was added and double arrowsindicate the time at which capsaicin was added.

FIGS. 4A-B depict Capsaicin induced neurotoxicity. 50B11 cells weregrown in 96-well plates and treated with varying doses of capsaicin (A)and capsaicin plus capsazepin (B) for 24 hours; cellular ATP levels weremeasured and expressed as a percentage of control cultures.(n=8/condition, error bars denote standard error of mean, *=p<0.05compared to controls)

FIGS. 5A-B depict ddC induced neurotoxicity and rescue by GPI-1046.50B11 cells were grown in 96-well plates and treated with varying dosesof ddC and ddC plus GPI-1046 (A) for 24 hours; cellular ATP levels weremeasured and expressed as a percentage of control cultures.(n=8/condition, error bars denote standard error of mean, *=p<0.05compared to controls). In validation experiments, 50B11 cells weredifferentiated and allowed to extend their neurites 24 hours. Then theywere treated with ddC or ddC plus GPI-1046 for another 24 hours, andtotal neurite lengths were measured (B). (n=6/condition, error barsdenote standard error of mean, *=p<0.05 compared to controls).

DETAILED DESCRIPTION OF THE INVENTION

To obtain immortalized cells, e.g., dorsal root ganglion cells, theinventors developed a method that reproducibly yields clonal lines ofcells, e.g., dorsal root ganglion cells, by introducing an oncogene andhTERT into the cells.

The methods of the invention are particularly useful in creatingimmortalized cells from cells that are known to be difficult toimmortalize. Specifically, the methods of the invention can be used withany cell type, but are particularly useful in cells that have beenhistorically difficult to immortalize, e.g., neuronal cells.

As used herein, the term “vector” refers to a polynucleotide constructdesigned for transduction/transfection of one or more cell typesincluding for example, “expression vectors” which are designed forexpression of a nucleotide sequence in a host cell, such as a neuronalcell.

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, refer to a polymeric form of nucleotides of any length, eitherribonucleotides or deoxyribonucleotides. These terms include a single-,double- or triple-stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid,or a polymer comprising purine and pyrimidine bases, or other natural,chemically, biochemically modified, non-natural or derivatizednucleotide bases. The backbone of the polynucleotide can comprise sugarsand phosphate groups (as may typically be found in RNA or DNA), ormodified or substituted sugar or phosphate groups. Alternatively, thebackbone of the polynucleotide can comprise a polymer of syntheticsubunits such as phosphoramidates and thus can be a oligodeoxynucleosidephosphoramidate (P—NH2) or a mixed phosphoramidate-phosphodiesteroligomer. Peyrottes et al. (1996) Nucleic Acids Res. 24: 1841-8;Chaturvedi et al. (1996) Nucleic Acids Res. 24: 2318-23; Schultz et al.(1996) Nucleic Acids Res. 24: 2966-73. A phosphorothioate linkage can beused in place of a phosphodiester linkage. Braun et al. (1988) J.Immunol. 141: 2084-9; Latimer et al. (1995) Molec. Immunol.32:1057-1064. In addition, a double-stranded polynucleotide can beobtained from the single stranded polynucleotide product of chemicalsynthesis either by synthesizing the complementary strand and annealingthe strands under appropriate conditions, or by synthesizing thecomplementary strand de novo using a DNA polymerase with an appropriateprimer.

The term “oncogene” as used herein is intended to mean a gene whoseaction promotes cell proliferation. Oncogenes are altered forms ofproto-oncogenes and are often expressed in cancerous cells. Exemplaryoncogenes include large T antigen, myc, abl, ras, and raf.

“Schwann cell” is a cell of neural crest origin that forms a continuousenvelope around each peripheral nerves fiber in situ. A Schwann cell canbe identified by detecting the presence of one or more markers ofSchwann cell such as glial fibrillar acidic protein (GFAP), proteinS100, laminin, or nerve growth factor (NGF) receptor, e.g., usingantibodies against these markers. Furthermore, Schwann cells have acharacteristic morphology that can be detected by microscopicexamination of cultures thereof.

“Immortalized cell line” as used herein means a cell line that canreplicate and be maintained indefinitely in in vitro cultures underconditions that promote growth, preferably at least over a period of ayear or years.

“Cell line” as used herein is a population or mixture of cells of commonorigin growing together after several passages in vitro. By growingtogether in the same medium and culture conditions, the cells of thecell line share the characteristics of generally similar growth rates,temperature, gas phase, nutritional and surface requirements. The cellline can become more homogenous with successive passages and selectionfor specific traits. Clonal cells are those which are descended from asingle cell. A cloned cell culture is a cell culture derived from asingle cell. Immortalized cell lines are cells that have been modifiedto undergo indefinite numbers of successive passages.

A SV40 Large T Antigen (SV-40 LTA) oncogene is intended to encompass anynucleotide sequence which encodes a protein having the function ofpolyoma (or SV-40) LTa and which is capable of being expressed in thehost cell, e.g., a neuronal cell.

The term “hTERT” as used herein is an abbreviation for the humantelomerase reverse transcriptase, i.e., the catalytic protein componentof human telomerase. hTERT is described in Cong, Y. S., et al. (1999)Hum. Mol. Genet. 8 (1), 137-142 and can be found in GenBank as AccessionNumber AAD12057. Although human TERT is exemplified herein, one of skillin the art will recognize that TERT molecules from other species, orvariants of human TERT that maintain the biological function of hTERTare useful in the methods of the invention.

The human telomerase catalytic subunit has been cloned (see Nakamura, etal. (1997) Science 277: 955; Mayerson, et al. (1997) Cell 90: 78; andKilian, et al. (1997) Hum Mol Genet 6: 2011; U.S. Pat. No. 6,166,178).Sources of the coding sequence for the human telomerase subunit includeany cells that demonstrate telomerase activity such as immortal celllines, tumor tissues, germ cells, proliferating stem or progenitorcells, and activated lymphocytes. The nucleic acid can be obtained usingmethods known in the art.

As used herein, the term “pain” is art recognized and includes a bodilysensation elicited by noxious chemical, mechanical, or thermal stimuli,in a subject, e.g., a mammal such as a human. The term “pain” includeschronic pain, such as lower back pain; pain due to arthritis, e.g.,osteoarthritis; joint pain, e.g., knee pain or carpal tunnel syndrome;myofascial pain, and neuropathic pain. The term “pain” further includesacute pain, such as pain associated with muscle strains and sprains;tooth pain; headaches; pain associated with surgery; or pain associatedwith various forms of tissue injury, e.g., inflammation, infection, andischemia.

As used herein, the term “pain disorder” includes a disease, disorder orcondition associated with or caused by pain. Examples of pain disordersinclude arthritis, allodynia, a typical trigeminal neuralgia, trigeminalneuralgia, somatoform disorder, hypoesthesis, hypealgesia, neuralgia,heuritis, neurogenic pain, analgesia, anesthesia dolorosa, causlagia,sciatic nerve pain disorder, degenerative joint disorder, fibromyalgia,visceral disease, chronic pain disorders, migraine/headache pain,chronic fatigue syndrome, complex regional pain syndrome,neurodystrophy, plantar fasciitis or pain associated with cancer.

The term pain disorder, as used herein, also includes conditions ordisorders which are secondary to disorders such as chronic pain and/orneuropathic pain, i.e., are influenced or caused by a disorder such aschronic pain and/or neuropathic pain. Examples of such conditionsinclude, vasodialation, and hypotension; conditions which arebehavioral, e.g., alcohol dependence (see, e.g., Hungund andBasavarajappa, (2000) Alcohol and Alcoholism 35:126-133); or conditionsin which detrimental effect(s) are the result of separate disorders orinjuries, e.g., spinal cord injuries.

The methods of the instant invention rely on the introduction of two DNAsegments, i.e., genes, into a cell which results in the formation of animmortalized cell. These cells can be further differentiated by theaddition of a differentiating agent. Importantly, the methods of theinvention rely on the incorporation of an oncogene and hTERT into acell, but the method of introducing these genes into the cell are ofsecondary importance.

The manner in which the oncogene or hTERT coding region is introducedinto the cells of interest is not critical, as long as a functionalpolypeptide is expressed. Expression can be extrachromosomal orfollowing integration into the cellular genome. Any of a variety oftechniques can be used to introduce the oncogene or the hTERT gene intothe desired cells, including electroporation, liposomes, or viralvectors. See Molecular Cloning, 3^(rd) Edition, 2001, by Sambrook andRussell. In one embodiment, the coding sequence is introduced using aviral vector, for example SV40, adenovirus, Herpes simplex virus,adeno-associated virus, and the like. See Blomer et al., Human MolecularGenetics 5 Spec No: 1397-404, 1996; Zern et al., Gene Ther. 6: 114-120,1999; and Robbins et al., Trends in Biotechnology 16: 35-40, 1998.

A specific means for incorporating the hTERT coding region into thecells of interest is to use a recombinant retrovirus that provides forintegratration of the DNA segment efficiently and stably into the genomeof the target cell.

The retroviral vectors generally include as operatively linkedcomponents, retroviral long terminal repeats, packaging sequences andcloning site(s) for insertion of heterologous sequences. Otheroperatively linked components may include a nonretroviralpromoter/enhancer and a selectable marker gene. Examples of retrovirusexpression vectors which can be used include DC-T5T (Sullenger et al.1990. Mol. Cell Biol. 10: 6512-65230), kat (Blood. 1994 83: 43-50), BOSC(Proc. Natl. Acad. Sci. (USA) (1993) 90: 8392-8396), pBabe (Proc. Natl.Acad. Sci. (USA) (1995) 92: 9146-9150) and RetroXpreSS.™. (Clontech,Palo Alto, Calif.). An expression vector is available that includes thehTERT gene, for example pBabe-puro-hTERT (Morgenstern and Land 1990). Insome instances, it may be desirable to increase expression of the hTERTgene by utilizing other promoters and/or enhancers in place of thepromoter and/or enhancers provided in the expression vector. Thesepromoters in combination with enhancers can be constitutive orregulatable. Any promoter/enhancer system functional in the target cellcan be used. (See for example, Molecular Virology pp. 176-177; Hofmann,et al. 1996. Proc. Natl. Acad. Sci. (USA) 93: 5185-5190; Coffin andVarmus, 1996. Retroviruses. Cold Spring Harbor Press, NY; Ausubel et al.1994. Current Protocols in Molecular Biology. Greene PublishingAssociates, Inc. & Wiley and Sons, Inc.). Examples include: CMVimmediate-early promoter, SV40, thymidine kinase promoter,metalothionine promoter, and tetracycline operator (Hofman et al.,(1996) Proc. Natl. Acad. Sci (USA) 93: 5185-5190).

For transfection, the neuronal cells or other cells to be transfectedare suspended in a suitable culture medium containing recombinantretrovirus vector particles. Many different suitable culture media arecommercially available. They include DMEM, IMDM, and .alpha.-MEM, with5-30% serum and often further supplemented with, e.g., BSA, one or moreantibiotics and optionally growth factors suitable for stimulating celldivision. Recombinant retrovirus vector particles are harvested intothis medium by incubating the virus-producing cells in this medium. Toenhance gene transfer, compounds such as polybrene, protamine sulphate,or protamine HCl generally are added. Usually, the cultures aremaintained for 24 days and the recombinant retrovirus vector containingmedium is refreshed daily. Optionally, the cells to be transfected areprecultured in medium with growth factors but without recombinantretrovirus vector particles for up to 2 days, before adding therecombinant retrovirus vector containing medium. For successful genetransfer it is essential that the target cells undergo replication inculture. It is often beneficial to transform, transfect, orelectroporate a number of times to obtain a higher number of cellscontaining the desired DNA segment. To maximize the number of cellscontaining the desired DNA segment, the cells are transformed,transfected, or electroporated and allowed to recover for a number ofhours or days and then transformed, transfected, or electroporatedagain. This process may be repeated 2, 3, 4, 5, 6 or more times in orderto maximize the number of cells containing the desired DNA. After thefinal cycle is performed, cells containing the desired DNA are selectedusing methods that are routine in the art.

In exemplified embodiments, the oncogene and hTERT are introduced intothe cell by electroporation. After one or more rounds ofelectroporation, cells containing the oncogene are selected using anantibiotic resistance marker introduced into the cell along with theoncogene. Once cells containing the oncogene are selected, hTERT isintroduced by electroporation. After one or more rounds ofelectroporation, cells containing hTERT are selected. The selected cellscontain both the oncogene and hTERT.

Once cells containing an oncogene and hTERT are selected, the cells canbe differentiated by exposing the cells to differentiation agent. In thecase of neuronal cells, this agent can be cAMP, a cyclic AMP analog, ora substance that increases intracellular levels of cAMP. Exemplary cAMPanalogs are 8-pCPT-2′-O-Me-cAMP, 8Br-cAMP, Sp-cAMPS, and forskolin. Inone exemplified embodiment, the agent is forskolin.

The immortalized cells produced by the methods described herein areparticularly useful in screening assays. Specifically, the cellsproduced by the methods of the instant invention are ideal for highthroughput screening. In a specific embodiments, the immortalizednociceptive DRG sensory neurons produced by the methods of the inventionare ideal for identifying modulators of neuropathic pain. Previousattempts at identifying modulators of neuropathic pain have focused onidentifying TRPV1 antagonists. However, TRPV1 has been expressed innon-neuronal cell lines and the calcium flux induced by capsaicin wasused as an outcome measure for high throughput screening (HTS)(Garcia-Martinez C et al. (2002) Proc Natl Acad Sci USA 99:2374-2379;Gunthorpe M J et al. (2004) Neuropharmacology 46:133-149; Masip I et al.(2004) J Comb Chem 6:135-141). Although these cells expressingrecombinant TRPV1 may be useful, a nociceptive sensory neuronal cellexpressing TRPV1 will be more relevant because the non-neuronal celllines may lack the appropriate intracellular signaling pathwaysassociated with and downstream of TRPV1 in nociceptive sensory neurons.Likewise, neuronal cells from other areas of the body can beimmortalized and would be useful for high throughput screening toidentify modulators for these cells. For example, sensory, motor orcortical neurons can be immortalized and used to identify modulators of,for example, Alzheimer's disease, Parkinson's disease, Huntington'sdisease, multiple sclerosis, or amyotrophic lateral sclerosis (ALS).

Accordingly, the instant invention provides methods of screening formodulators of human cells, e.g., human neuronal cells, by contacting animmortalized cell of the invention with a candidate modulator anddetermining if the modulator has a desired biological effect, e.g.,binding to and/or modulating the activity of TRPV1.

In a specific embodiment, the invention provides screening methods usingthe immortalized nociceptive DRG neurons produced by the methods of theinvention to identify modulators of pain, e.g., neuropathic pain.Immortalized nociceptive DRG neurons are contacted with candidatemodulators and the ability to modulate, for example, capsaicin inducedtoxicity can be monitored to determine if a candidate modulator is amodulator of neuropathic pain.

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., peptides, peptidomimetics, small molecules, ribozymes,or antisense molecules) which bind to bind to and/or modulate theactivity of the immortalized cells of the invention. Compoundsidentified using the assays described herein may be useful for treatingpain disorders.

Candidate/test compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam, K. S. et al. (1991) Nature354:82-84; Houghten, R. et al. (1991) Nature 354:84-86) andcombinatorial chemistry-derived molecular libraries made of D- and/orL-configuration amino acids; 2) phosphopeptides (e.g., members of randomand partially degenerate, directed phosphopeptide libraries, see, e.g.,Songyang, Z. et al. (1993) Cell 72:767-778); 3) antibodies (e.g.,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and singlechain antibodies as well as Fab, F(ab′)₂, Fab expression libraryfragments, and epitope-binding fragments of antibodies); and 4) smallorganic and inorganic molecules (e.g., molecules obtained fromcombinatorial and natural product libraries).

Candidate modulators can be obtained using any of the numerousapproaches in combinatorial library methods known in the art, including:biological libraries; spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to polypeptide libraries, while the otherfour approaches are applicable to polypeptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, K. S. (1997) Anticancer DrugDes. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andin Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries of compoundsmay be presented in solution (e.g., Houghten (1992) Biotechniques13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor(1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409),spores (Ladner U.S. Pat. No. '409), plasmids (Cull et al. (1992) Proc.Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990)Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla etal. (1990) Proc. Natl. Acad. Sci. 97:6378-6382); (Felici (1991) J. Mol.Biol. 222:301-310.

Alternatively, test compounds can be designed based on the structure ofknown modulators of pain, or compounds that are know to bind toreceptors expressed by neurons.

The ability of a given modulating agent to modulate pain can bequantitated by using any one of the following tests: tight ligation ofL6 and L7, as a model of neuropathic pain; complete Freund's adjuvantinto knee joint or hind paw as a model of Long term inflammatory pain(Palecek, J. (1992) Neurophysiol 68:1951-66); nerve ligation (CCI);thermal hyperalgesia, tactile allodynia and cold allodynia (Carlton, S.M. et al. (1994) Pain 56:155-66); thermal paw withdrawal latency(Hargreaves test); von Frey mechanical withdrawal threshold; thehot-plate latency test; the tail flick test (Stone, L. S., et al. (1997)NeruroReport 8:3131-3135); the warm-water immersion tail flick assay(Stone, L. S., et al. (1997) NeruroReport 8:3131-3135); the crush injuryto the sciatic nerve test (De Konig, et al. (1986) J. Neurol. Sci.74:237-246); the cold water allodynia test (Hunter, et al. (1997) Pain69:317-322; the paw pressure latency assay (Hakki-Onen, S., et al.(2001) Brain Research 900(2):261-7; or the radiant heat test (Yoshimura,M., (2001) Pharm. Research 44(2):105-11).

Briefly, the tail flick latency test involves projecting a beam of lightto the tail of an animal. The time is measured from the onset of thetail heating and stops at the moment of the tail flick. Typically, fivetail flick latency (TFL) measurements are made per rat per session with5-10 minutes between trials.

The preceding paragraphs set forth high throughput screening methodsusing immortalized nociceptive DRG neurons to identify modulators ofneuropathic pain. However, one of skill in the art could adapt theseassays to identify modulators of other conditions and immortalized celllines made using the methods of the invention.

EXAMPLES

It should be appreciated that the invention should not be construed tobe limited to the examples that are now described; rather, the inventionshould be construed to include any and all applications provided hereinand all equivalent variations within the skill of the ordinary artisan.

Example 1 Generation and Characterization of DRG Neuronal Cell Line

Materials and Methods

Unless noted otherwise all reagents and materials were purchased fromInvitrogen (Carlsbad, Calif.). Animal studies were conducted accordingthe protocols approved by the institutional Animal Care and UseCommittee.

Construction of SV40 Large T-Antigen and hTERT Expression Vectors

For cloning of the SV40 large T-antigen, plasmid construct pZipSV776-1was used as the template for PCR amplification of the gene fragmentcoding for SV40 large T-antigen. PCR reaction was primed byoligonucleotides 5′-CACCGCTTTGCAAAGATGGATAAAG (sense) and5′-AATTGCATTCATTTTATGTTTCA (anti-sense). Amplification was performedusing the Expend High Fidelity PCR System (Roche, Indianapolis, Ind.).The PCR product was cloned into the pENTR/D-TOPO vector by directionalTA-cloning. After confirmation of the sequence, the target SV40 largeT-antigen gene was transferred into pLenti6/V5-Dest vector using Gatewaytechnology. In the destination vector, the SV-40 large T-antigen wasunder the control of P_(cmv), and the selection marker, blasticidinresistance gene, was under the control of P_(sv40). The hTERT expressionconstruct pBabe-hygro-hTERT carrying hygromycin resistant gene (also akind gift of William C. Hahn at Harvard University), was used totransfer the hTERT gene into the pLenti6/V5-Dest vector using Gatewaytechnology. In the destination vector, the hTERT was under the controlof P_(cmv), and the selection marker, hygromycin resistance gene, wasunder the control of P_(sv40). The expression plasmids were prepared andpurified using Plasmid MIDI Kit (Qiagen, Valencia, Calif.).Endotoxin-free plasmid was suspended in distilled water forelectroporation.

Electroporation into Dissociated DRG Neurons and Selection of Clones

Dissociated primary DRG neuronal cells were prepared as previouslydescribed (Hoke A et al. (2003) J Neurosci 23:561-567; Keswani S C etal. (2003) Ann Neurol 53:57-64) and the plasmid was electroporated.Approximately 5×10⁴ cells in 90 ml Opti-MEM media were mixed with 10 mlplasmid (1 mg/ml) and transferred into a 0.2 cm Gene Pulser cuvette(Bio-Rad, Hercules, Calif.). After 10 minutes of incubation at roomtemperature, a single square-wave pulse (100 V, 950 mF, ˜40 ms) wasdelivered by a Gene Pulser II with a Capacitance Extender Plus (Bio-Rad,Hercules, Calif.). Culture medium at 4° C. was immediately added to thecells and the cuvette was kept on ice for 10 minutes. Cells were platedin T75 flasks in culture medium without antibiotics (Neurobasal medium,10% FBS, 0.5 mM glutamine, 1×B-27 supplement, 0.2% glucose). In order toincrease the efficiency of electroporation and incorporation of largeT-antigen into terminally differentiated sensory neurons, the process ofelectroporation was repeated 3-4 times before addition of antibioticselection media. About 60-70% of the cells survived the electroporationprocess. Twenty-four hours after the last electroporation, culturemedium was replaced by selection medium containing blasticidin (5 μg/ml)and cells were maintained in this medium for 1-2 weeks until isolatedcolonies with 200-300 cells formed. Colonies were picked and expandedusing standard culture methods when reached 80-90% confluence. For hTERTtransduction, SV40 transfected and blasticidin resistant cells weretrypsinized and electroporated with the hTERT plasmid as above for thelarge T-antigen. The electroporation was repeated 34 times. About 60-70%of the cells survived the electroporation process. Twenty-four hoursafter the last electroporation, culture medium was replaced by selectionmedium containing hygromycin (50 mg/ml) and cells were maintained inthis medium for 1-2 weeks until isolated colonies with 200-300 cellsformed. Colonies were picked and expanded using standard culture methodswhen reached 80-90% confluence.

Induction of Neuronal Differentiation and Characterization of theImmortalized Neuronal Clone

One of the immortalized DRG neuronal cell lines (50B11) maintainedself-replication capability over many cell divisions (>300) and was usedin further analysis of neuronal properties. Differentiation and axonalelongation was induced in these cells by addition of forskolin (50 μM)into the culture medium. Within hours, more than 90% cells stoppeddividing and extended long neurites. These cells were grown in 24-wellplates on glass coverslips, fixed with 4% paraformaldehyde andimmunostained for presence of neurofilament (SMI-32 antibody fromSternberger Monoclonals Inc., Lutherville, Md.), βIII-tubulin (Promega,Madison Wis.), transient receptor potential channel, vanilloid subfamilymember-1 (TRPV-1) (Abcam, Cambridge, Mass.), calcitonin gene relatedprotein (CGRP) (source, city, state) or isolectin B4 (IB4) (VectorLaboratories, Burlingame, Calif.) using standard methods (Keswani etal., supra). Slides were counterstained with4′,6-Diamidino-2-phenylindole (DAPI) and mounted with Vectashield(Vector Laboratories, Burlingame, Calif.). Specificity of all primaryand secondary antibodies was confirmed using appropriate positive andnegative control cultures.

The electrophysiological recording techniques employed were similar tothose described by Hamill et al. (Hamill O P et al. (1981) Pflugers Arch391:85-100). The external solution contained (mM) 145 NaCl, 5 KCl, 2CaCl₂, 1 MgCl₂, 10 D-glucose and 10N-2-hydroxyethylpiperazine-Nρ-2-ethanesulfonic acid (HEPES) (pH 7.4;310-320 mOsmol.) Cells were continuously superfused at 2-3 ml/min. Usingthe whole-cell patch-clamp technique, data were obtained withborosilicate thin-walled micropipettes (BORO, BF150-110-10, Sutter,Novato, Calif.) made with a Flaming-Brown Puller (P-87, SutterInstruments, Novato, Calif.). Micropipettes were filled with (in mM) 140KCL, 1 CaCl₂, 1 MgCl₂, 10 HEPES, 10 ethyl glycol-bis(3-aminoethylether)-N,N,Nρ,Nρ-tetraacetic acid (EGTA), 4 Mg-ATP adjusted to a pH of7.3 with Tris buffer. Pipette resistances measured 3 to 6 MΣ.Current-clamp recordings were obtained with an Axopatch 200B amplifier(Axon Instruments Inc. Foster City, Calif.) and data was filteredon-line at 2 kHz. Recordings were made at a holding potential (V_(H)) of−60 mV. For statistical evaluation we used ANOVA (Origin version 6,Microcal Software Inc., Northampton, Mass.)

For analysis of changes in gene expression, 50B11 cells were grown inmedia containing forskolin (50 μM), NGF (10 ng/ml), GDNF (10 ng/ml) orvehicle control for 24 hours. Total RNA was isolated using the TRIzolReagent according to the manufacturer's recommendation. Two μg total RNAwas reverse-transcribed using Ready-To-Go You-Prime First-Strand Bead(Amersham Biosciences, Piscataway, N.J.) according to manufacturer'sprotocols. Real-time PCR was carried out in a DNA Engine OpticonContinuous Fluorescence Detection System using DyNAmo SYBR GreenPolymerase (MJ Research, Waltham, Mass.). All primers were designedaccording to the individual gene sequence in the GenBank/EMBL nucleotidesequence database (primer sequences are available upon request). Thebinding positions of all primers were chosen to produce amplicons of 150to 200 base pairs and to achieve maximum efficiency and specificity. Allprimer sequences were checked for specificity by a BLAST search in theGenBank/EMBL nucleotide sequence database. The primers were synthesizedby Integrated DNA Technologies, Inc. (Coralville, Iowa). Theamplification of internal control housekeeping gene, GAPDH, was carriedout using a commercial kit (Applied Biosystems, Foster City, Calif.)according to the protocol supplied by the manufacturer. The expressionlevels of individual genes before and after treatment were calculatedusing the comparative C_(T) method.

Ca²⁺ microfluorimetry and imaging in forskolin-differentiated 50HB cellswere performed by ratiometric imaging of the Ca2+-sensitive fluorescentdye fura-2. Cells were grown on glass coverslips in 12-well dishes andcalcium imaging was done with and without differentiation with 50-μMforskolin. Cell were loaded for 15 min at 37° C. with 2 M fura-2acetoxymethyl ester (Molecular Probes, Carlsbad, Calif.) in Krebs-HEPESbuffer (100 mM NaCl, 2.0 mM KCl, 1.0 mM CaCl2, 1.0 mM MgCl2, 1.0 mMNaH2PO4, 4.2 mM NaHCO3, 12.5 mM HEPES and 10.0 mM glucose), then washedfor 3 times in buffer to remove remaining fura-2 ester. The coverslipwith loaded cells was then mounted on an inverted epifluorescencemicroscope (Zeiss, Axiovert 200) and covered with 60 μl Krebs-HEPESbuffer or buffer with capsaicin. In capsazepin pretreatment experiments,capsazepin was added Images were acquired every ˜3 seconds with anextended Hamamatsu Digital Camera C4742-95 (Hamamatsu Photonics,Barcelona, Spain) using a dual filter wheel (Sutter Instruments, Novato,Calif., USA) equipped with 340 and 380 nm, 10-nm-bandpass filters (OmegaOptics, Madrid, Spain). Data was acquired using InCyt Im2 software(Intracellular Imaging, Inc.). Fluorescence changes are expressed as theratio of fluorescence at 340 and 380 nm (F₃₄₀/F₃₈₀).

Neuronal Toxicity Assays

Conditions for culturing the 50B11 cells and measuring the ATP levelswere optimized for the 96-well plate format. Initially 500 cells/wellwere plated in 96-well plates for 24 hours and then differentiated withforskolin (50 μM) in culture medium with reduced serum (0.2%). Varyingconcentrations of ddC with or without immunophilin ligand GPI-1046 wereadded to the wells for another 24 hours. Cellular ATP levels weremeasured using the ViaLight Plus kit (Cambrex, city, state) according tomanufacturer's instructions. This luciferase-based assay allowsmeasurement of ATP levels on a luminometer with minimal manipulation ofthe well contents.

Measurements of axonal lengths to determine axonal degeneration inducedby ddC were done as described before (Keswani et al., supra). Briefly,DRG cultures were prepared from embryonic E14.5 rats, plated ontocollagen coated glass coverslips and allowed to extend axons for 48hours. Then ddC, GPI-1046 or vehicle controls were added to the mediafor another 24 hours. Cells were fixed and stained withanti-βIII-tubulin antibody to delineate the axons. Axon lengthmeasurements were done in multiple fields using a random samplingmethod. Each experiment was done in triplicates and repeated at leasttwice. Statistical analysis was done using ANOVA with correction formultiple comparisons.

Results

Immortalized DRG Neuronal Cells Extend Neurites Express Neuronal Markersand Generate Action Potentials after Differentiation

One of the clones generated after immortalization was further studiedafter evaluation using the initial screening of neurite extension inresponse to forskolin. This clone, 50B11, stopped dividing immediatelyafter addition of forskolin and within 4 hours extended neurites atleast twice as long as the neuronal body diameter (FIGS. 1A and 1B).Within 24 hours of differentiation, the cells were positive for neuronalmarkers βIII-tubulin and neurofilament (FIGS. 1C and 1D). We studiedthese cells before and after differentiation using patch clamping. Datawere obtained from a total of 14 cells (8 undifferentiated cells and 6differentiated cells). Collectively these cells displayed a mean restingmembrane potential of −57.9±2.1 mV. There were no statisticallysignificant differences between the mean resting potential values of theundifferentiated and differentiated groups (−57.4±1.9 mV versus−58.3±2.7 mV, mean±SEM; ANOVA, P>0.05). No spontaneous activity, eithersynaptic or action potential discharge, was observed when differentiatedor undifferentiated cells were held at their resting membrane potentialfor periods up to 10 min. Electrical stimulation of undifferentiatedcells with depolarizing current steps did not induce an action potential(n=0/8). On the other hand, when differentiated cells were stimulated,action potentials could be elicited (n=5/6; FIG. 1E).

50B11 Neuronal Line Express Markers of Small Diameter Sensory Neurons

Small diameter DRG sensory neurons are generally divided into twocategories; peptidergic ones with dependence on NGF, and non-peptidergicones with dependence on GDNF (Bennett D L et al. (1998) J Neurosci18:3059-3072). Markers such as CGRP for peptidergic neurons and IB4 fornon-peptidergic neurons can identify these subgroups of small diametersensory neurons. The 50B11 line expressed both markers whendifferentiated in the presence of forskolin (FIGS. 2A and 2B).Furthermore, 50B11 cells expressed receptors for NGF (low affinity NGFreceptor p75 and high affinity Trk-A) and GDNF (c-ret and GDNF familyreceptor alpha-1, GFRα-1) and upregulated these receptors whendifferentiated with forskolin (FIG. 2C). Interestingly, the upregulationof receptors was neurotrophic factor specific; NGF receptor, Trk-A wasmore upregulated in the presence of NGF compared to GDNF and similarlyGDNF receptor, GFRα-1 was more upregulated in the presence of GDNFcompared to NGF.

50B11 Neuronal Line Express Nociceptive Markers and Respond to Capsaicin

Once we determined that the 50B11 line had small diameter sensoryneuronal markers, we explored the possibility that it was a nociceptiveneuron. The cells expressed capsaicin receptor TRPV-1 and upregulatedtheir expression when differentiated with forskolin with and withoutneurotrophic factor treatment (FIG. 3). Furthermore, the 50B11 cellsresponded to capsaicin with a rapid rise in intracellular calciumlevels. This effect of capsaicin on the 50B11 cells was preventable bypretreatment of the cells with capsazepin, a specific blocker of TRPV-1,suggesting that the effect of capsaicin was mediated through TRPV-1. Agraph representative of multiple intracellular calcium measurements isshown FIG. 3C.

Next, we tested whether we can evaluate the neurotoxicity of capsaicinin an assay suitable for 96-well plate format. Capsaicin causes axonaldegeneration and death of nociceptive DRG sensory neurons [ref here]. Inorder to evaluate cell survival we used a luciferase-based assay tomeasure cellular ATP levels. We first optimized the assay by measuringATP levels in different numbers of cells grown in the 96-well plates andfound that the optimum number of cells for neurotoxicity assays wasbetween 500 and 1000 cells per well. We also optimized the cultureconditions and found that low serum levels of 0.2% fetal bovine serumprovided the most reliable results (data not shown). We then examinedthe dose-response curve of capsaicin and found that 10 μM of capsaicincaused about 50% reduction in ATP levels (FIG. 4A); similar to the doserequired for neurotoxicity of capsaicin in primary DRG neurons. Thecapsaicin-induced neurotoxicity was preventable by co-administration ofTRPV-1 blocker capsazepin in a dose-dependent manner (FIG. 4B).

The Immortalized 50B11 Neuronal Line is Suitable for High-ThroughoutDrug Screening

One of the potential uses of 50B11 sensory neuronal line will be theiruse in high-throughput screening assays. Our laboratory had developed invitro model of antiretroviral toxic neuropathy using primary DRG sensoryneurons (Keswani et al., supra). We adapted this assay to the 50B11cells and measured cellular ATP levels after varying concentrations ofddC (FIG. 5A). There was a dose-dependent toxicity of ddC atconcentrations similar to therapeutic plasma levels in HIV patients.This neurotoxicity was preventable by co-administration of aneuroprotective compound, GPI-1046, a non-immunosuppressive immunophilinligand. We also validated this toxicity using a more standard measure ofaxonal degeneration where we differentiated the 50B11 cells, let themextend neurites and then treated them with ddC with and without GPI-1046for 24 hours and then measured their total neuritic lengths (FIG. 5B).

Discussion

Cellular tools for drug development for peripheral neuropathies andneuropathic pain are limited. We developed a novel method to immortalizenociceptive dorsal root ganglion (DRG) neurons and show that theimmortalized DRG neuronal line (50B11) extend neurites whendifferentiated, express receptors characteristic of small sensoryneurons and nociceptive receptor TRPV-1, generate action potentials whendepolarized and respond to capsaicin. Furthermore, the cells are easy togrow in large quantities and suitable for high throughput drug screeningusing in vitro assays for neuropathic pain and peripheral neuropathies.

DRG neurons are terminally differentiated cells that extend long axonsto their target tissues. More than half of all DRG neurons areunmyelinated, extend axons to the skin and have nociceptive properties.In many studies on peripheral neuropathies and neuropathic pain, rat ormouse primary DRG neurons are used. However, obtaining these cells insufficient quantities to perform high-throughput drug screening isnearly impossible. An alternative is to use neuronal cell lines derivedfrom neuroblastomas or immortalized neural crest precursor/stem celllines (Rao M S and Anderson D J (1997) J Neurobiol 32:722-746). However,these approaches have caveats for neuropathic pain research and drugscreening. These cells are often heterogeneous and retain the potentialto differentiate into multiple neuronal and non-neuronal cell types in amixed environment. Furthermore, they are not likely to respond to drugsin a consistent manner because of this heterogeneity in culture. Incontrast, the nociceptive DRG neuronal cell line that we developed isclonal; therefore, all of the cells are similar to each other and inbiological assays behave in a predictable manner. These cells can begrown in large quantities, differentiated into nociceptive neurons thatexpress proper markers and ion channels necessary for nociception andgeneration of action potentials, and used in high-throughput drugscreening.

The immortalized DRG neuronal line, 50B11, was likely to be generatedfrom a nociceptive neuron with potential to differentiate into either apeptidergic neuron with NGF dependency or non-peptidergic neuron withGDNF dependency. During development, all future nociceptive neurons aredependent on NGF and express markers for NGF-dependent neurons (p75 andTrkA) (McMahon S B et al. (1994) Neuron 12:1161-1171), but a subgroupswitch dependency and express markers of GDNF-dependent neurons such asIB4 (Molliver D C et al. (1997) Neuron 19:849-861). The 50B11 lineretained this bi-potentiality because in the presence of a givenneurotrophic factor, it upregulated the appropriate receptors.Furthermore, the cells extended longer neurites in the presence ofeither NGF or GDNF.

In order to immortalize rat DRG neurons, we used a two-steptransformation process. Although combination of SV-40 large T antigenand hTERT had been used before to immortalize primary airway epithelialcells (Lundberg A S et al. (2002) Oncogene 21:4577-4586), this approachhad not been applied to generation of immortalized cell lines fromterminally differentiated cells such as neurons. In order to increaseour transfection efficiency and generation of transformed neurons, weused a different approach and performed multiple electroporations beforeadding the selection antibiotic to the media. We obtained multipleclones and characterized one in detail. We were able to grow this cellline, 50B11, through multiple doublings (well over 300) without loss ofdifferentiation potential. Stocks of cells from early and late passageshad similar properties in terms of their differentiation potential.

Differentiation into neurons with nociceptive properties wasaccomplished by using forskolin. During development DRG neurons expresshigh levels of cAMP but downregulate it after they are mature and theiraxons reach the target tissues. Elevated cAMP levels in the DRG linecould be accomplished by methods other than forskolin, but we choseforskolin mainly because of ease of use and relatively lower costcompared to other choices such as membrane permeable dibutryl-cAMP(db-cAMP). This is an important issue to consider in designing assaysfor high-throughput screening. Furthermore, we chose an assay that isalso easy to scale up for high-throughput screening. Measurement ofcellular ATP levels using the luciferase-based assay is suitable inmultiple ways. First, one can use it as a measure of cellular health andcell numbers as ATP levels in cells correlate with the number of healthycells. Second, the assay is simple to administer with no wash stepsinvolved. It requires addition of reagents into the wells twice, once tolyse the cells and second to add reagents for the luminescence. Thisimproves reproducibility of the assay and will reduce the number ofmultiplicates needed during drug screening.

In summary we have developed an immortalized DRG sensory neuronal linewith nociceptive properties suitable for high-throughput drug screeningfor peripheral neuropathies and neuropathic pain. The transfection andselection methods we developed can be used to generate other neuronalpopulations, including neurons from human tissues such as brain, spinalcord, dorsal root sensory ganglia or autonomic ganglia.

Example 2 Generation and Characterization of Human Fetal Astrocytes andSchwann Cells

Human astrocytes or Schwann cells were prepared from aborted fetaltissues using standard cell culture techniques. Cells were cultured inNeurobasal media (Neurobasal MEM plus 10% FBS, 0.5 uM glutamin, 2%glucose, 1×B27 supplement) for 3-7 days in an incubator with 5% CO2 at37° C. Cells were detached with a cell scraper and washed 2 times inOpti-MEM (Invitrogen, CA) and 0.8-1.0×10⁶ cells were suspended in 100 ulOpti-MEM and dissociated with pipetting using a 200 μl tip.

Five μg pLenti6/V5-DEST plasmid carrying hTERT gene (pLenti6/hTERT) in1.7 μl TE (pH8.0) and 15 μg pLenti6/V5-DEST plasmid carrying SV40 largeT antigen gene (pLenti6/SV40) in 5 μl TE (pH8.0) were mixed with cells,transferred into a 0.2 cm gene-pulser cuvette (Bio-Rad, CA) andincubated for 5 minutes at room temperature. Gene Pulser XcellElectroporation System (Bio-Rad, CA) was set at 850 μF×90V and the cellswere pulsed once (with a time constant of 3540 mini-second). 500 μlice-chilled culture media (antibiotics free) was immediately added tothe cells and the cuvette was kept on ice for 5 minutes. Cells were thentransferred into a 75 cm² (T75) culture flask and cultured inantibiotics-free Neurobasal media for 3-7 days until 70-80% confluence.

The next 3 electroporations were done with the same procedure, but withdifferent amount of pLenti6/hTERT and pLenti6/SV40. For the secondelectroporation, 10 μg pLenti6/hTERT (in 3.3 μl TE, pH 8.0) and 10 μgpLenti6/SV40 (in 3.3 μl TE, pH 8.0) were mixed with cells. For the thirdtime electroporation, 15 μg pLenti6/hTERT (in 5 μl TE, pH8.0) and 5 μgpLenti6/SV40 (in 1.7 μl TE, pH8.0) were mixed with cells. For the fourthelectroporation, 20 μg pLenti6/hTERT (in 6.6 μl TE, pH8.0) and 2 μgpLenti6/SV40 (in 0.6 μl TE, pH8.0) were mixed with cells.

After the fourth electroporation, cells from each gene-pulser cuvettewere transferred into 3 T75 culture flasks and cultured in Neurobasalmedia containing 5 μg/ml blasticidin (Invitrogen, CA) for 6-8 days.Blasticidin-resistant colonies were detached with 0.05% trypsin,isolated with glass capillary pipettes and transferred into 24 wellplates. Cells were then cultured for 5-10 days (depending on the size ofthe original colony) in blasticidin-containing media and a portion ofthe cells were plated in a T75 culture flask for further cloning. Theresulted colonies were propagated in T75 flasks, stored frozen infreezing media (culture media plus 10% DMSO), and characterized.

The immortalized human astrocyte and Schwann cell lines were furthercharacterized by RT-PCR, immunohistochemistry and Western blotting. Celllines expressed glial markers such as GFAP, S100, CD44 and tenascin byRT-PCR, and GFAP and S100 by immunohistochemistry. Furthermore,astrocyte line expressed glutamate transporters EAAT-1 and EAAT-2 byRT-PCR and western blotting. Further characterization, includingbiological assays are ongoing.

INCORPORATION BY REFERENCE

The contents of all references, patents, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method for generating an immortalized human cell comprising:introducing into a cell a DNA segment encoding an oncogene; selectingfor a cell containing the DNA segment; and introducing a hTERT into theselected cell; thereby generating an immortalized cell.
 2. The method ofclaim 1, wherein the DNA segment is contained in a plasmid.
 3. Themethod of claim 1, wherein hTERT is introduced by a retrovirus.
 4. Themethod of claim 1, wherein the cell is a neuronal cell.
 5. The method ofclaim 4, wherein the neuronal cell is selected from neuronal cells fromthe brain, spinal cord, and dorsal root sensory ganglia.
 6. The methodof claim 1, wherein the human cell is a dorsal root ganglia neuron. 7.The method of claim 6, wherein the dorsal root ganglion neuron is anociceptive dorsal root ganglion neuron.
 8. The method of claim 1,wherein the cell is a glial cell.
 9. The method of claim 8, wherein theglial cell is an astrocyte, oligodendrocyte or a Schwann cell.
 10. Themethod of claim 1, further comprises contacting the immortalized cellwith an agent that causes differentiation.
 11. The method of claim 10,wherein the agent is cyclic AMP or an analog thereof or an agent thatincreases intracellular cAMP levels.
 12. The method of claim 11, whereinthe agent is forskolin.
 13. A method of producing immortalized neuronalcells comprising: introducing a DNA segment encoding an oncogene into aneuronal cell; selecting for neuronal cells that contain the DNAsegment; introducing hTERT into the selected cells; and selecting forcells that contain hTERT; thereby producing immortalized neuronal cells.14. The method of claim 13, wherein the DNA segment is contained in aplasmid.
 15. The method of claim 13, wherein the neuronal cells areselected from a group consisting of neuronal cells from the brain,neuronal cells from the spinal cord, dorsal root sensory ganglia, andautonomic ganglia.
 16. The method of claim 13, wherein the oncogene isselected from the group consisting of Ras, Myc, Raf, and largeT-Antigen. 17-28. (canceled)
 29. A method of producing immortalizeddorsal root ganglion neuronal cell line comprising: introducing aplasmid comprising an SV40 large T-antigen into dorsal root ganglioncell; selecting dorsal root ganglion cells that contain the plasmid;introducing hTERT into the selected cells; and selecting for cells thatcontain hTERT; thereby producing immortalized dorsal root ganglionneuronal line.
 30. The method of claim 29, wherein hTERT is introducedby a retrovirus.
 31. An immortalized nociceptive dorsal root ganglionneuron.
 32. The immortalized nociceptive dorsal root ganglion neuron ofclaim 31, wherein the neuron expresses markers of nociceptive dorsalroot ganglion neurons.
 33. The method of claim 24, wherein thenociceptive dorsal root ganglion neurons express a capsaicin receptor,TRPV1, GDNF-receptor, NGF-receptor, or a sodium channel.
 34. The methodof claim 24, wherein the nociceptive dorsal root ganglion neuronsrespond to capsaicin by elevating intracellular calcium flux or generateaction potentials when polarized.
 35. The immortalized nociceptivedorsal root ganglion neuron of claim 33, wherein the capsaicin receptoris TRPV1.
 36. The immortalized nociceptive dorsal root ganglion neuronof claim 35, further comprising an oncogene and hTERT. 37-40. (canceled)41. A method of identifying a modulator of pain, comprising: contactingan immortalized dorsal root ganglion neuronal cell of claim 29, with acandidate modulator; and determining if the candidate modulator is bindsto and/or modulates the dorsal root ganglion neuronal cell; therebyidentifying a modulator of pain.